Rapid diagnostic method for distinguishing allergies and infections and nasal secretion collection unit

ABSTRACT

A method and device for rapidly, non-invasively and inexpensively differentiating between allergic rhinitis, upper respiratory tract viral infection and bacterial sinusitis, comprising a support strip upon which is fixed discrete indicators of pH, protein content, nitrite content, leukocyte esterase activity, and eosinophil content or other measure of a substance found in allergic secretions, such as TAME esterase, of a sample with which said reagent test strip is contacted. Contact of a nasal secretion with the device of this invention permits differentiation between allergic, bacterial and viral conditions, based on pH, protein content, leukocyte esterase activity, nitrite content, eosinophil content and TAME esterase activity. The invention further provides a novel means for collecting nasal secretions to facilitate differential diagnosis of sinusitis, upper respiratory tract viral infection and allergic rhinitis.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/535,699 filed Jan. 9, 2004, which is a continuation-in-part ofapplication Ser. No. 10/015,520, filed Dec. 13, 2001, which is adivisional of U.S. application Ser. No. 09/597,360, filed Jun. 19, 2000,which is a continuation in part of PCT application serial numberPCT/US99/05751 filed Mar. 16, 1999, which in turn was acontinuation-in-part of application Ser. No. 08/621,557 Mar. 25, 1996,now U.S. Pat. No. 5,910,421, which was a continuation in part ofapplication Ser. No. 08/576,604, filed on Dec. 21, 1995, now abandoned.

BACKGROUND OF THE INVENTION

I. Field of the Invention

This invention is a rapid and simple method for the differentialdiagnosis of allergies, sinusitis and upper respiratory tractinfections. The method involves the use of either commercially availableor novel, specifically adapted, indicator or reagent test strips whichare contacted with nasal secretions. Based on the differential read-outfrom the indicator strip, and a measure of eosinophil infiltration orother substance in the nasal secretion, a user of the strip is able todetermine, with the assistance of a scoring method disclosed herein,whether an allergic condition, a viral infection or bacterial sinusitisis the cause of the respiratory discomfort. In particular, thisinvention provides an improved device for providing differentialdiagnosis whereby a patient may easily deposit nasal secretions on theindicator elements by blowing their nose into a container, such as abag, in which the indicator device may be inserted, or in which theindicator element forms an integral part.

II. Background

It is common for patients afflicted with respiratory discomfort to seekthe advice of a clinician in an effort to minimize or overcome theirdiscomfort. Such discomfort generally is attributable to one of thefollowing etiologies: allergic reactions, viral upper respiratory tractinfections (URIs), or bacterial infections which can produce sinusitis.However, the clinician presented with such a patient typically has thedaunting task of determining which of these three principal etiologiesis responsible for the discomfort experienced by a particular patient.The danger inherent in a mis-diagnosis can be quite severe. For example,should the clinician incorrectly diagnose an allergy as sinusitis, acourse of antibiotics would typically be prescribed. Naturally, suchtreatment would do little to alleviate the allergic discomfort beingexperienced by the patient while at the same time, the patient isexposed to an antibiotic to which there is a possibility of raising aresistant bacterial infection. Should this occur, a problem much moresevere than the original allergic condition will have been unwittinglyengendered. The prevalence of antibiotic-resistant strains on a globalscale due to the over-prescribing of antibiotics has become anincreasingly recognized problem (Service, R. F., 1995).

In the foregoing example, the availability of a rapid and simpledifferential diagnostic method would, instead of resulting in acompounded problem, result in the simple recommendation by the clinicianthat the patient adhere to a course of antihistamine treatment, allergenavoidance, and/or a regimen of toleragenic desensitization.Unfortunately, however, to date, there is no such simple procedure whichwill provide the clinician with the necessary differential diagnosis. Anaccepted method of diagnosis for bacterial sinusitis is expensiveradiologic imaging (typically X-ray or CT-scan) of the patients' sinuses(see Katz et al., 1995).

Many scientific articles have appeared addressing one or another of thevarious etiologies of respiratory discomfort. However, no rapid,inexpensive differential diagnostic method has been found. Thus, forexample, Wang et al., Correlations between Complaints, InflammatoryCells and Mediator Concentrations in Nasal Secretions after NasalAllergen Challenge and during Natural Allergen Exposure, Int. Arch.Allergy Immunology 1995; 106:278-285, disclosed a method of using anasal microsuction technique. They showed that nasal allergen challenge(NAC) of asymptomatic (out of season) seasonal allergic rhinitispatients results in immediate (5 minutes) increases in histamine,leukotriene C4 (LTC4), and tryptase, with a more gradual (one hour postNAC) and prolonged increase in eosinophil and eosinophil cationicprotein (ECP) concentration in nasal secretions. By contrast, insymptomatic (in season allergic rhinitis) patients, high concentrationsof eosinophils, ECP, LTC4 and histamine, but not tryptase, wereobserved. It was concluded that allergic rhinitis is a chronicinflammation of the nasal mucosa, and that infiltration of eosinophilsand release of late-phase inflammatory mediators are the predominantpathophysiologic markers. However, this publication neither teaches norsuggests that these observations can be applied to distinguish patientssuffering from an allergic condition as opposed to an infection. Inaddition, the methods used by these authors are laborious andtime-consuming and do not involve the use of reagent test strips. Sigurset al., Eosinophil cationic protein in nasal secretion and in serum andmyeloperoxidase in serum in respiratory syncytial virus bronchiolitis:relation to asthma and atopy, Acta Paediatr 1994; 83:1151-5, concludedthat it is not possible to predict, from eosinophil cationicprotein/albumin ratios in nasal secretions or from ECP andmyeloperoxidase concentrations in serum, whether children withrespiratory syncytial virus (RSV) bronchiolitis would develop asthma.The publication provides no teaching or suggestion of a method which caneasily distinguish patients suffering from an allergic condition asopposed to an infection. The methods used by these authors are laboriousand time-consuming and do not involve the use of reagent test strips.Okuda et al., A Novel Method of Counting Eosinophils in Nasal Secretionof Allergic Rhinitis by Hemocytometric Method, Int. Arch. AllergyImmunol. 1994; 104 (suppl. 1):6, disclosed a rapid method forquantifying the number of eosinophils in nasal secretions as a methodfor diagnosis of allergic rhinitis. The method involves preparation of asolubilized sample of nasal secretion and counting of whole eosinophils.There is no mention of reagent test strips and there is no mention of amethod for distinguishing patients suffering from an allergic conditionas opposed to an infection.

Kowalski et al., Neutrophil chemotactic activity (NCA) in nasalsecretions from atopic and non-atopic subjects, Allergy 1993;48:409-414, reported that basal nasal secretions of both healthy personsand patients with chronic rhinitis contain significant chemotacticactivity to neutrophils. The study also reports that there is anincrease in protein content of nasal secretions in patients withperennial allergic rhinitis (AR) following challenge with an antigen.There is no mention of reagent test strips and there is no mention of amethod for distinguishing patients suffering from an allergic conditionas opposed to an infection.

Igarashi et al., Analysis of nasal secretions during experimentalrhinovirus upper respiratory infections, J. Allergy Clin. Immunol. 1993;92:722-731, studied patients with allergic rhinitis or control subjectsinoculated with rhinovirus. Nasal lavage samples pre- and post-infectionwere analyzed for protein and mast cell mediators. It was found thattotal protein (including the plasma proteins albumin and IgG and theglandular proteins lactoferrin, lysozyme and secretory IgA) increasedpost-infection, predominantly due to increased vascular permeability. Itwas also found that the allergic subjects had fewer symptoms, butgreater vascular permeability and greater histamine secretion thancontrol subjects post-rhinovirus infection. Protein was determined bythe bicinchoninic acid protein assay (Pierce Chemical Co.) on aliquotsof nasal lavage. It is noted at several points in the publication thatthe symptoms of rhinovirus infected patients and patients with nasalallergic reactions are similar, thus teaching away from the possibilitythat a simple nasal secretion assay method could be used to distinguishthese conditions. Inasmuch as this study is directed at determining thedifferences in nasal secretions between rhinovirus infected normal orallergic individuals, the study addresses a different problem than thataddressed by the instant invention, which is a method for measuring thedifferences in nasal secretions of patients infected with a rhinovirus,for example, and a patient not infected with a rhinovirus but sufferingfrom allergic rhinitis. The methods used by these authors are laboriousand time-consuming and do not involve the use of reagent test strips.

Sperber et al., In vivo detection of a novel macrophage-derived proteininvolved in the regulation of nasal mucus-like glycoconjugate secretion,J. Allergy Clin. Immunol. 1993; 92:581-588, disclosed a study directedat characterization of a novel 68 Kd nasal mucus secretagogue (NMS-68)released by monocytes. Inasmuch as this protein was found to be presentin nasal tissue of patients with allergic and non-allergic rhinitis, itdoes not appear to provide a method for distinguishing between theseconditions, although the baseline level of this protein is more elevatedin the allergic patients. Reagent test strips were not used in thisstudy.

Knani et al., Indirect evidence of nasal inflammation assessed bytitration of inflammatory mediators and enumeration of cells in nasalsecretions of patients with chronic rhinitis, J. Allergy Clin. Immunol.1992; 90:880-889, examined the nasal lavage cells and six inflammatorymediators released in nasal secretions of four groups of patients withperennial rhinitis and a control group. It was found that patients withsymptomatic allergic rhinitis had increased levels of eosinophils, aswell as of eosinophil protein X (EPX), LTC4/D4, tryptase, MPO and PGD2.Patients with non-allergic rhinitis were found to have increasedneutrophil, tryptase, MPO and EPX concentrations. These measurementsprovided indirect evidence of nasal inflammation. However, there was noanalysis of differences between the nasal secretions of rhinitispatients and patients suffering, for example, from a rhinoviralinfection or a bacterial sinus infection.

Klementsson et al., Eosinophils, secretory responsiveness andglucocorticoid-induced effects on the nasal mucosa during a weak pollenseason, Clinical and Experimental Allergy 1991; 21:705-710, analyzed theeosinophil influx, the concentration of ECP and secretory responsivenessfollowing methacholine challenge in nasal lavage samples of patientswith allergic rhinitis. There was no concurrent analysis of nasalsecretions from patients with bacterial sinus or viral infections, andreagent test strips were not employed.

Gordon et al., The pathophysiology of rhinitis, J. Allergy Clin.Immunol. 1991; 88:33-42, challenged patients with seasonal rhinitis onone side of the nose with an allergen and nasal secretions from bothsides of the nose were analyzed for protein and mediators. There was noconcurrent analysis of nasal secretions from patients with bacterial orviral infections, and reagent test strips were not employed.

Cohen, R. A, and Brestel, E. P., Nasal secretory response to allergenprovocation, 1988; 18:435-443, analyzed nasal lavage samples fromragweed-sensitive and control subjects following ragweed pollenchallenge. The study revealed no increase in total protein, albumin,potassium, lysozyme activity or peroxidase activity in the controlsubjects. There were increases in all of these constituents in theragweed-sensitive subjects. These constituents were assayed by dyebinding (Bradford), rocket immunoelectrophoresis, absorptionspectroscopy, ABTS (Sigma Chemical Co.) oxidation, and radial diffusion,respectively. Use of reagent test strips is neither taught norsuggested, nor is there a concomitant analysis of these constituents inbacterially or viral infected, no-allergic subjects.

Liu et al., Injurious effect of eosinophil extract on the human nasalmucosa, Rhinology 1988; 26:121-132, attempted to elucidate the role ofeosinophils in nasal secretions of allergic subjects. These authorsconclude that the eosinophil extracts tested may actually be harmful tothe function of human nasal mucosa. Use of reagent test strips isneither taught nor suggested, nor is there a concomitant analysis ofthese constituents in infected subjects.

Anderson et al., Allergen-induced nasal hyperactivity appears unrelatedto the size of the nasal and dermal immediate allergic reaction, Allergy1987. 42:631-637, analyzed “priming”, in which nasal lavage samples fromhay fever patients were tested following an initial and a re-challengewith allergen. The biochemical parameter used as the measure of theallergic reaction was TAME-esterase via a radiochemical method (releaseof tritium labeled methanol from the synthetic substrate H3-TAME). Useof reagent test strips is neither taught nor suggested, nor is there aconcomitant analysis of these constituents in infected subjects.

Settipane, G. A, and Klein, D. E., Non Allergic Rhinitis: Demography ofEosinophils in Nasal Smear, Blood Total Eosinophil Counts and IgELevels, NER Allergy Proc. 1985; 6:363-366, in an attempt to develop amethodology for differential diagnosis of patients with non-allergicrhinitis, evaluated patients with rhinitis and negative skin tests,(taken to mean that their rhinitis had a non-allergic etiology), forcause of the rhinitis. Nasal smears from these patients were obtained byrolling a swab with nasal secretions on a glass slide, fixing withmethanol, staining with Camaco stain (Wright-Giemsa stain), and countingthe number of eosinophils per 100 cells. Sinus X-rays were conducted todetect sinusitis. NARES, non-allergic rhinitis with eosinophiliasyndrome, is clinically defined in the paper as “nasalcongestion/rhinorrhea with negative allergy skin tests, normal serumIgE, and ≧5% eosinophils in the nasal smear.” The purpose of the studywas “to attempt to corroborate NARES as a new syndrome and to attempt tofurther classify and clarify non-allergic rhinitis.” There was nodiscussion in this paper regarding the problem of mis-identification ofthis clinical condition with upper respiratory tract infections, and useof reagent test strips as part of the differential diagnosis is neitherdisclosed nor suggested.

Brofeldt, et al., Biochemical Analysis of Nasal Secretions induced byMethacholine, Histamine, and Allergen Provocations, Am. Rev. Respir.Dis. 1986; 133:1138-1142, obtained methacholine, histamine and allergeninduced nasal secretions from subjects over a fifteen minutepost-induction period. The nasal secretions were weighed and tested forhexose content (orcinol method), protein content (Lowry method),carbohydrate (gas liquid chromatography), sialic acid (calorimetricthiobarbituric acid assay), inorganic sulphate (radioactive BaCl2), DNA(diphenylamine), albumin and immunoglobulins (rocketimmunoelectrophoresis or ELISA). The different inducers were found tohave differential effects on the various elements tested. However, therewas no concomitant study of nasal secretions from patients sufferingfrom an infection nor was the use of reagent test strips taught orsuggested.

Eggelston, et al., Mediators of Immediate Hypersensitivity in NasalSecretions during Natural Colds and Rhinovirus Infection ActaOtolaryngol. 1984; suppl. 413:25-35, note that “[v]iral respiratoryinfections and allergic rhinitis have many similarities. Not only aresymptoms similar in the two conditions, but the pathologic anatomy ofboth is dominated by vascular dilatation and edema with minimal cellularinfiltrate in acute phases . . . .” The authors postulated that thesesimilarities are due to mast cell activation during infection, resultingin release of histamine. However, they report that spectrofluorometricanalysis of histamine in nasal secretions of control subjects orpatients with a natural cold or with a rhinovirus infection does notsupport this hypothesis. Histamine concentrations were found to begenerally lower in individuals infected with influenza A or rhinovirus.TAME-esterase was also found not to be elevated during these infections.This article reports that during viral respiratory tract infection thereis little or no TAME-esterase elevation, while in allergic rhinitisthere is TAME esterase elevation (see discussion at page 34 of thereference). However, the use of reagent test strips for this purpose isneither taught nor suggested, nor is there a discussion of how theseresults could be used in a differential diagnostic method also aimed atdistinguishing sinusitis.

Baumgarten, et al, Plasma Kallikrein During Experimentally InducedAllergic Rhinitis: Role in Kinin Formation and Contribution toTAME-Esterase Activity in Nasal Secretions, J. Immunol. 1986;137:977-982, report the observation that when allergic and non-allergicindividuals are challenged intranasally with an allergen, post-challengenasal lavages of only the allergic individuals contained elevated levelsof immunoreactive human plasma kallikrein/prekallikrein (iHPK). Thisincrease in iHPK correlated with increases in kinins, histamine,TAME-esterase, and clinical symptoms. In fact, these researchers arguethat the TAME-esterase activity is produced by plasma kallikrein andmast cell tryptase. Thus, iHPK may be an additional marker that could beused in a reagent test strip for allergic rhinitis diagnosis. However,in this study, esterase was assayed by a radiochemical method and kininswere assayed using a radioimmunoassay, rather than by any type ofreagent test strip.

Anderson et al., Mechanisms of nasal hyper-reactivity, Eur. Arch.Otorhinolaryngol. 1995; 252 (suppl. 1):S22-S26, review the factors knownto be involved in allergen-induced nasal hyper-reactivity. However,there is no teaching or suggestion of a method for distinguishingallergic from infection related rhinitis. Reagent test strips for thispurpose are neither proposed nor suggested.

Florman, et al., Rapid Non-invasive Techniques for Determining Etiologyof Bronchitis and Pneumonia in Infants and Children, Clin. Chest Med.1987; 8:669-679, provide a review of techniques for differentialdiagnosis of the causative agent in lower respiratory tract infections.A number of rapid, non-specific and specific tests are mentioned.However, there is no mention of differential diagnosis of allergic frominfective conditions and reagent test strips for this purpose areneither disclosed nor suggested.

Katz et al., A comparison of Imaging Techniques in Patients with ChronicSinusitis (X-Ray, MRI, A-Mode Ultrasound)Allergy Proc. 1995. 16:123-127,demonstrate the long-felt need for a rapid, inexpensive way to diagnosesinusitis and distinguish this condition from allergic rhinitis. Themethod of the instant invention has the potential for supplanting themuch more expensive diagnostic techniques reported in this paper to bemost reliable in diagnosis of sinusitis (CAT scans and MRI).

Demoly et al., Assessment of Inflammation in noninfectious chronicmaxillary sinusitis, J. Allergy Clin. Immunol. 1994; 94:95-108, suggestthat it might be possible to distinguish sinusitis (infection) fromallergic rhinitis based on the contents of nasal mucosa. However, thetechniques used in attempting to distinguish these conditions dependedon the use of immunohistochemistry of surgical specimens,immunocytochemistry of lavage fluids, and measurement of specificinflammatory mediators in sinus lavage fluids (ELISA, RIA). There is noteaching or suggestion that reagent dip-sticks could be used for thispurpose.

Accordingly, there has been a long-felt need in the art for a rapid,inexpensive, non-invasive technique for a method capable ofdistinguishing between allergies and infections. The method of theinstant invention involves testing nasal secretions with commerciallyavailable (Ames Division, Miles Laboratories, Inc., Elkhart, Ind. 46515,or Boehringer Mannheim Corporation, Advanced Diagnostics, 9115 HagueRoad, P.O. Box 50457, Indianapolis, Ind. 46250-0457) or novel ormodified reagent test strips. The commercially available strips, alsoreferred to as dipsticks, test for pH, protein, glucose, ketone, whiteblood cell esterase, bilirubin and blood.

The following U.S. patents which may be relevant to the instantinvention are listed on the product insert of the Miles LaboratoriesInc., reagent test strips, and are herein incorporated by reference:

-   1) U.S. Pat. No. 3,438,737—Protein test composition and method of    detecting proteins in fluids using the test composition.-   2) U.S. Pat. No. 4,301,115—Test device having resistance to cross    contamination between reactant areas and process for making it.-   3) U.S. Pat. No. 4,637,979—Composition and test device for    determining the presence of leukocytes containing a zwitterion    coupling agent for determining the presence of leukocytes, esterase    or protease in a sample.-   4) U.S. Pat. No. 4,645,842—Pyrrole composition for detecting    presence of hydrolytic analytes, useful in the detection of    leukocytes, esterase and protease in a test sample.-   5) U.S. Pat. No. 4,657,855—Composition and test device for    determining the presence of leukocytes, esterase and protease in a    test sample.-   6) U.S. Pat. No. 4,704,460—Novel compounds for detecting the    presence of hydrolytic analytes in a test sample which indicates the    presence of leukocytes, esterase and protease in a test sample.-   7) U.S. Pat. No. 4,758,508—Analytical process and agents for the    detection of esterolytic and/or proteolytic enzymes in a liquid    sample.

The following U.S. patents which may be relevant to the instantinvention are listed on the product insert or packaging of BoehringerMannheim Corporation's reagent test strips, and are herein incorporatedby reference:

-   1) U.S. Pat. No. 3,359,072—A protein determination method.-   2) U.S. Pat. No. 3,418,079—A protein determination device and    method.-   3) U.S. Pat. No. 3,712,853—A nitrite detection reagent and method.-   4) U.S. Pat. No. 3,897,214—A diagnostic device.-   5) U.S. Pat. No. 3,802,842—A reagent test strip.-   6) U.S. Pat. No. 4,013,416—A protein detection method.-   7) U.S. Pat. No. 4,385,114—An oxidation indicator system.

However, none of these patents disclose or suggest a method for testingnasal mucous secretions to distinguish allergic from infectiousconditions.

In the method of this invention, a sample of a patient's nasalsecretions is tested and, based on the pH, amount of protein, nitriteand leukocyte esterase, it can quickly be determined if the patient issuffering from bacterial sinusitis. The secretions can also be testedfor eosinophils, eosinophil components, TAME esterase, and if present insufficient amounts, a diagnosis of allergy can be made. Viral infectionscan be deduced by the absence of allergy or sinusitis. The method hasthe potential to supplant much more expensive and invasive clinicalprocedures. In addition, the present invention provides an improvementto that which is disclosed in application Ser. No. 08/576,605, now U.S.Pat. No. 5,910,421, of which the present disclosure is acontinuation-in-part. The improvement provided herein is a collectiondevice for home or point of use care for the collection of nasalsecretions and conducting diagnosis therewith out end user contact withthe sample. That is, the present invention disclosure provides for aself-contained collection device. Features which may be incorporatedinto various embodiments of this device include air vents, foldablesealing means, patient identification means, and the like, as disclosedin further detail herein below.

BRIEF SUMMARY OF THE INVENTION

This invention provides a method for rapidly, non-invasively andinexpensively differentiating between allergic rhinitis and respiratoryviral or bacterial infections. The method involves measuring, forexample, by contacting a reagent test strip with a sample of nasalsecretion, a series of agents in nasal secretions. The reagent teststrip provides information on the pH, protein content, nitrite content,leukocyte esterase activity and preferably also provides information onthe level of eosinophil infiltration in the sample contacted, such thata combination of a pH between about 7.5 and 9, a moderately strongpresence of protein, at least a trace of leukocyte esterase and nitrite(see scoring system below) indicates the presence of a bacterialinfection (sinusitis). The combination of a pH between about 5.0 and7.5, little or no protein, little or no leukocyte esterase activity andlittle or no eosinophil infiltration or TAME esterase activity, is anindication of viral upper respiratory infection (URI). However, the sameprofile as in URI viral infection but with clear indication ofeosinophil infiltration, TAME esterase activity or both is an indicationof allergic rhinitis. As noted above, Eggelston et al., and Baumgartenet al., have noted that TAME-esterase is not elevated during influenza Aor rhinoviral infections, while in allergic rhinitis, there is esteraseelevation. Accordingly, in one embodiment of the present invention, achromogenic substrate, such as Tosyl-Arg-paranitrophenol ester orparanitroaniline, Z-Arg-paranitroaniline (Bachem, Cat. #L1220), aderivative of Tosyl-Arg-paranitro phenol ester designed to liberate3-hydroxy-5-phenyl pyrrole, which may then be reacted with a diazoniumsalt, B-z-Arg-paranitroaniline, or like colorimetric reagents orTAME-like substances or substrates, are included as diagnosticdiscriminators for allergic rhinitis. Development of color on contact ofthis reagent with nasal secretion therefore would be indicative ofallergic rhinitis.

Also provided is a reagent test strip specifically adapted for rapidly,non-invasively and inexpensively differentiating between allergy andrespiratory infection. The reagent test strip is adapted to provideinformation on the pH, protein content, nitrite content, leukocyteesterase activity, eosinophil infiltration and TAME esterase activity ofthe sample contacted, such that the method of this invention can quicklyand easily be practiced.

In addition, this invention provides a practical, simple and safecollection device for collection of nasal secretions. The collectiondevice may contain integral indicators for providing the differentialdiagnosis, or the collection device may be adapted such that a separateindicator device may be used to provide the differential diagnosis basedon the nasal secretions collected therein.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 shows the different chemical read-outs obtained by contactingreagent test strips with the nasal secretions of multiple patientspresenting with respiratory discomfort. Closed circles (●) representpatients with radiologically proven sinusitis; open circles (◯)represent patients having no sinusitis based on radiologicalinvestigation; open squares (□) represent patients with radiologicallyproven sinusitis that were at least partially treated with antibioticsprior to or during the evaluation period.

FIG. 2 is a histogram based on the data shown in FIG. 1. From the rawdata of FIG. 1, a point system was established such that; pHs greaterthan or equal to 8.5 received a score of +3, a pH between 8.0 and 8.4received a score of +2, a pH between 7.25 and 7.9 received a score of +1and a pH between 5.0 and 7.25 received a score of zero; protein contentbetween about 30-100 mg/dl was given a score of +1; between about 100mg/dl and 300 mg/dl was given a score of +2; protein between about 300mg/dl and 2000 mg/dl was given a score of +3; and protein greater than2000 mg/dl received a score of +4; low nitrite concentrations received ascore of +1; moderate amounts of nitrite received a score of +2 and highamounts of nitrite received a score of +3; leukocyte esterase activityin small amounts received a score of about +1, a score of +2 wasassigned moderate amounts and a score of +3 was assigned to largeamounts of leukocyte esterase. By summing the scores assigned to pH,nitrite, protein, and leukocyte esterase, the histogram of FIG. 2 wasproduced. Patients radiologically confirmed to have sinusitis are shownwith dark bars; patients radiologically clear of sinusitis are shown byopen bars.

FIG. 3 provides a graphic representation of the novel test kit andcollection apparatus of this device, as well as an illustration of themethod of use thereof.

Accordingly, it is an object of this invention to provide a safe, simpleand self-contained device for providing differential diagnosis betweensinusitis, viral respiratory tract infection, and allergic rhinitis.

A further object of this invention is to provide a novel nasal secretioncollection device.

DETAILED DESCRIPTION OF THE INVENTION

This invention is a method for non-invasively, rapidly and simplydistinguishing between allergies and infections which involves testingnasal secretions for the levels of a number of agents. Preferably themethod comprises contacting nasal secretions with commercially available(for example, from Ames Division, Miles Laboratories, Inc., Elkhart,Ind. 46515, or from Boehringer Mannheim Corporation, AdvancedDiagnostics, 9115 Hague Road, P.O. Box 50457, Indianapolis, Ind.46250-0457) or novel specifically adapted reagent test strips. Thecommercially available strips, also referred to as dipsticks, typicallytest for pH, protein, nitrite glucose, ketone, white blood cellesterase, bilirubin and blood.

In the method of this invention, a sample of a patient's nasalsecretions is contacted with a reagent test-strip and, based on the pH,presence or absence of protein, levels of nitrite, leukocyte esteraseand a measure of eosinophil infiltration in the nasal secretion, it canquickly be determined if the patient is suffering from an allergicreaction, a viral infection or a bacterial sinusitis infection. Themethod has the potential to supplant much more expensive and invasiveclinical procedures. In the method of this invention, a commerciallyavailable reagent test-strip, or a novel, specifically adapted reagenttest-strip, is contacted with a sample of a patient's nasal secretions.The patient may be any mammal, including an animal or a human. Based onthe differential read-out from the reagent test-strip, which preferablyincludes a measure of eosinophil infiltration, TAME esterase activity orboth, a determination can be made as to whether an allergy, sinusitis ora viral infection is responsible for upper-respiratory tract discomfort.With respect to commercially available reagent test-strips, those soldby the Ames Division of Miles Laboratories or the Boehringer MannheimCorporation are generally acceptable. These test-strips may be made asdisclosed herein or as disclosed in any of U.S. Pat. Nos. 3,438,737,4,301,115; 4,637,979. 4,645,842; 4,657,855; 4,704,460; 4,758,508;3,359,072; 3,418,079. 3,712,853; 3,897,214; 3,802,842; 4,013,416; and4,385,114; all of which are incorporated by reference herein for thispurpose. These reagent test-strips may be employed in the novel methodof this invention as further described hereinbelow with the additionalrequirement being that a measure of eosinophils or other substancecharacteristic of allergic secretions, such as TAME esterase activity,must be made.

Accordingly, one embodiment of this invention is a simple method fordistinguishing patients with allergies and patients with upperrespiratory infections involves testing the nasal secretions of thesepatients with reagent strips disclosed in the aforementioned patents.These dipsticks are inexpensive, and provide an indication of acontacted fluid's pH, protein, glucose, ketone, nitrite, leukocyteesterase, bilirubin and blood content. The patient blows his/her nose onor into a receptacle (for example wax paper or plastic film such asSaran Wrap or the like, or into a collection device specifically adaptedfor this purpose, as further disclosed herein below), or a swab of nasalsecretion is taken, and contacted with the test strip. The pH, protein,nitrite, and leukocyte esterase contents are then evaluated based on thedirections of the manufacturer found on the outside of the box in whichthe commercially available reagent test strips are sold. The proceduregenerally takes between about sixty seconds to about two minutes. Thefollowing table provides results and the meaning thereof:

Bacterial Infection Allergy Viral Infection (Sinusitis) pH 5.0-7.5 7.5-97.5-9 Protein Trace to + Trace to + ++ or +++ Nitrite Negative* or traceNegative or trace ++ or +++ Leukocyte Negative* or trace Trace to + ++or +++ Esterase *Note: “Negative” in this context means a non-positivetest result, rather than that a mathematically “negative” score isassigned to this quantity (i.e. no detectable presence of the item beingmeasured).

This data can be visualized by referring to FIG. 1 which provides agraphic representation of the different chemical read-outs obtained bycontacting reagent test strips with the nasal secretions of multiplepatients presenting with respiratory discomfort. Closed circles (●)represent patients with radiologically proven sinusitis; open circles(◯) represent patients having no sinusitis based on radiologicalinvestigation; open squares (□) represent patients with radiologicallyproven sinusitis that were at least partially treated with antibioticsprior to or during the evaluation period. As can be seen from FIG. 1,there is a clustering in the data points for the pH, protein, nitriteand leukocyte esterase contents of patients with or without sinusitis.

To aid in the differential diagnosis of allergic rhinitis, viral URI andsinusitis, we have discovered that by assigning a point system toseveral parameters measured from the mucus secretions of the patients,delimited differential scattering of the patient populations can beachieved with only a minor amount of overlap between patients havingthese various clinical conditions. FIG. 2 is a histogram based on thedata shown in FIG. 1. From the raw data of FIG. 1, a point system wasestablished such that; pHs greater than or equal to 8.5 received a scoreof +3, a pH between 8.0 and 8.4 received a score of +2, a pH between7.25 and 7.90 received a score of +1 and a pH between 5.0 and 7.25received a score of zero; protein content between about 30-100 mg/dl wasgiven a score of +1; between about 100 mg/dl and 300 mg/dl was given ascore of +2; protein between about 300 mg/dl and 2000 mg/dl was given ascore of +3 and protein greater than 2000 mg/dl received a score of +4;low nitrite concentrations received a score of +1; moderate nitriteconcentrations received a score of +2; high concentrations of nitritereceived a score of +3; leukocyte esterase activity in small amountsreceived a score of about +1, a score of +2 was assigned to moderateamounts and a score of +3 was assigned to large amounts of leukocyteesterase. By summing the scores of the pH, nitrite, protein, andleukocyte esterase, the histogram of FIG. 2 was produced. Patientsradiologically confirmed to have sinusitis are shown with dark bars;patients radiologically clear of sinusitis are shown by open bars. Thismethod of differential diagnosis can be used with any method ofcollecting the pH, protein, leukocyte esterase, and nitrite, and is notrestricted to the use of reagent indicator strips. The use of reagentindicator strips is, however, one of the most easily conducted,inexpensive and rapid methods for achieving this analysis.

The level of eosinophil infiltration is easily estimated in oneembodiment of the invention using high power field microscopy (about400× magnification) by determining the percentage of total cells thatare eosinophils in a Hansel's stained smear of nasal secretion.

According to the package insert of the AMES 9SG Multistix® product, thefollowing information, supplied for urinalysis by that manufacturer, maybe directly applied to the novel utility disclosed and claimed herein,and is hereby incorporated as follows:

REAGENT STRIPS for mucus analysis are firm plastic strips to which areaffixed several separate reagent areas. Depending on the product beingused, AMES REAGENT STRIPS provide tests for glucose, bilirubin, ketone(acetoacetic acid), specific gravity, blood, pH, protein, urobilinogen,nitrite, and leukocytes in urine. A user of these strips refers to thecarton and bottle label for specific reagent areas on the product beingused.

The reagent test areas on AMES REAGENT STRIPS are ready to use uponremoval from the bottle and the entire reagent strip is disposable. Thestrips may be read visually, requiring no additional laboratoryequipment for testing. Certain configurations of strips may also be readinstrumentally, using the CLINITEK® family of Urine Chemistry Analyzersand the appropriate Program Module or Program Card from AMES.

The directions must be followed exactly. Accurate timing is essential toprovide optimal results. The reagent strips must be kept in the bottlewith the cap tightly closed to maintain reagent reactivity. We havesurprisingly found that in six samples tested immediately aftercollection of nasal secretions or six hours after collection andrefrigeration, identical results were obtained. Through this finding,those skilled in the art may conclude that the method disclosed hereinmay be practiced immediately after collection of nasal secretions, ormay be stored for later measurement.

Chemical Principles of the Procedure:

pH: This test is based on a double indicator principle that gives abroad range of colors covering the entire urinary pH range. Colors rangefrom orange through yellow and green to blue.

Protein: This test is based on the protein-error-of-indicatorsprinciple. At a constant pH, the development of any green color is dueto the presence of protein. Colors range from yellow for “Negative”through yellow-green and green to green-blue for “Positive” reactions.

Nitrite: At the acid pH of the reagent area, nitrite in the nasalsecretion reacts with p-arsanilic acid to form a diazonium compound.This diazonium compound in turn couples with1,2,3,4-tetrahydrobenzo(h)quinolin-3-ol to produce a pink color. Theintensity of the pink color developed is used as the basis for assigningthe nitrite concentration score as described above.

Leukocytes: Granulocytic leukocytes contain esterases that catalyze thehydrolysis of the derivatized pyrrole amino acid ester to liberate3-hydroxy-5-phenyl pyrrole. This pyrrole then reacts with a diazoniumsalt to produce a purple product. The intensity of the purple colordeveloped is used to assign a value to esterase activity as describedabove.

TAME esterase: As noted above, Eggelston et al., and Baumgarten et al.,have noted that TAME-esterase is not elevated during influenza A orrhinoviral infections, while in allergic rhinitis, there is esteraseelevation. Accordingly, in one embodiment of the present invention, achromogenic substrate, such as Tosyl-Arg-paranitrophenol ester orparanitroaniline, Z-Arg-paranitroaniline (Bachem, Cat. #L1220), aderivative of Tosyl-Arg-paranitro phenol ester designed to liberate3-hydroxy-5-phenyl pyrrole, which may then be reacted with a diazoniumsalt, or like colorimetric reagents are included as diagnosticdiscriminators for allergic rhinitis. Development of color on contact ofthis reagent with nasal secretion therefore would be indicative ofallergic rhinitis.

Reagents (Based on Dry Weight at Time of Impregnation):

pH: 0.2% w/w methyl red; 2.8% w/w methyl red; 2.8% w/w bromthymol blue;97.0% w/w non-reactive ingredients.

Protein: 0.3% w/w tetrabromphenol blue; 97.3% w/w buffer; 2.4% w/wnonreactive ingredients.

Nitrite: 1.4% w/w p-arsanilic acid; 1.3% w/w1,2,3,4-tetrahydrobenzo-(h)-quinolin-3-ol; 10.8% w/w buffer; 86.5% w/wnonreactive ingredients.

Leukocytes: 0.4% w/w derivatized pyrrole amino acid ester; 0.2% w/wdiazonium salt; 40.9% w/w buffer; 58.5% w/w nonreactive ingredients.

Those skilled in the art will recognize that other chemical componentsmay be used for carrying out the method disclosed herein.

Recommended Procedures for Handling Reagent Strips: All unused stripsmust remain in the original bottle. Transfer to any other container maycause reagent strips to deteriorate and become unreactive. Do not removedesiccant(s) from bottle. Do not remove strip from the bottle untilimmediately before it is to be used for testing. Replace cap immediatelyand tightly after removing reagent strip. Do not touch test areas of thereagent strip. Work areas and specimen containers should be free ofdetergents and other contaminating substances. Dip test areas in mucuscompletely, but briefly, to avoid dissolving out the reagents. If usingstrips visually, read test results carefully at the times specified, ina good light (such as fluorescent) and with the test area held near theappropriate Color Chart on the bottle label. Do not read the strips indirect sunlight. If the strips are used instrumentally, carefully followthe directions given in the appropriate instrument operating manual.Protection against ambient moisture, light and heat is essential toguard against altered reagent.

Discoloration or darkening of reagent areas may indicate deterioration.If this is evident, or if test results are questionable or inconsistentwith expected finding, the following steps are recommended: (1) confirmthat the product is within the expiration date shown on the label; (2)check performance against known positive control materials (e.g.,CHEK-STIX® Control Strips); (3) retest with fresh product.

The following procedure should be followed exactly to achieve reliabletest results:

-   1. Collect fresh mucus specimen on a non-absorbent surface.-   2. Remove one strip from the bottle and replace cap. Completely    immerse reagent areas of the strip in fresh mucus and remove    immediately to avoid dissolving out reagents.-   3. If reading visually, compare reagent areas to corresponding Color    Chart on the bottle label at the time specified. Hold strip close to    color blocks and match carefully. Avoid laying the strip directly on    the Color Chart, as this will result in soiling the chart.-   4. If reading instrumentally, carefully follow the directions given    in the appropriate instrument operating manual.

Proper read time is critical for optimal results. If using stripsvisually, read the pH, protein, and nitrite at 60 seconds; andleukocytes at 2 minutes. The pH and protein areas may also be readimmediately or at any time up to 2 minutes after dipping.

After dipping the strip, check the pH area. If the color on the pad isnot uniform, read the reagent area immediately, comparing the darkestcolor to the appropriate Color Chart. All reagent areas exceptleukocytes may be read at about 1 minute for identifying negativespecimens and for determination of the pH. A positive reaction (small orgreater) at less than 2 minutes on the leukocyte test may be regarded apositive indication of leukocytes. Color changes that occur after 2minutes are of no diagnostic value. If using strips instrumentally, theinstrument will automatically read each reagent area at a specifiedtime.

For best results, performance of reagent strips should be confirmed bytesting known negative and positive specimens or controls whenever a newbottle is first opened. Negative and positive specimens or controls mayalso be randomly hidden in each batch of specimens tested. Eachlaboratory should establish its own goals for adequate standards ofperformance, and should question handling and testing procedures ifthese standards are not met. CHEK-STIX® Urinalysis Control Strips(#1360) from AMES®, with positive or defined results, provide aconvenient basis for quality control program.

Results with AMES REAGENT STRIPS are obtained in clinically meaningfulunits directly from the Color Chart comparison when using stripsvisually. With instrumental use, the reagent pads are “read” by theinstrument and the results are displayed or printed. The color blocksand instrumental display values represent nominal values; actual valueswill vary around the nominal values.

pH: If proper procedure is not followed and excess mucus remains on thestrip, a phenomenon known as “runover” may occur, in which the acidbuffer from the protein reagent will run onto the pH area, causing afalse lowering of the pH result.

Protein: Contamination of the nasal secretion specimen with quaternaryammonium compounds (e.g., from some antiseptics and detergents) or withskin cleansers containing chlorhexidine may also produce false positiveresults.

Nitrite: Pink spots or pink edges should not be interpreted as apositive result. Any degree of uniform pink color development should beinterpreted as a positive nitrite test.

Leukocytes: Elevated glucose concentrations (≧3 g/dl) or high specificgravity may cause decreased test results. The presence of cephalexin(Keflex®), cephalothin (Keflin®), or high concentrations of oxalic acidmay also cause decreased test results. Tetracycline may cause decreasedreactivity, and high levels of the drug may cause a false negativereaction.

Leukocytes: Normal nasal secretion will generally yield negativeresults; positive results (small or greater) are clinically significant.Individually observed trace results may be of questionable clinicalsignificance; however, trace results observed repeatedly may beclinically significant. Positive and repeated trace results indicate theneed for further testing of the patient and/or nasal specimen, accordingto medically accepted procedures.

Specific Performance Characteristics: Specific performancecharacteristics are based on clinical and analytical studies. Inclinical specimens, the sensitivity depends upon several factors: thevariability of color perception; the presence or absence of inhibitoryfactors, the specific gravity, and the pH; and the lighting conditionswhen the product is read visually. Because the color of each reagentarea changes as the analyte concentration increases, the percentage ofspecimens detected as positive will increase with the analyteconcentration.

Each color block or instrumental display value represents a range ofvalues. Because of specimen and reading variability, specimens withanalyte concentrations that fall between nominal levels may give resultsat either level. Exact agreement between visual results and instrumentalresults might not be found because of the inherent differences betweenthe perception of the human eye and the optical system of theinstruments.

pH: The pH test area measure pH values generally to within 1 unit in therange of 5-8.5 visually and 5-9 instrumentally.

Protein: The reagent area is more sensitive to albumin than toglobulins, hemoglobin, Bence-Jones Protein and mucoprotein; a negativeresult does not rule out the presence of these other proteins.

Nitrite: Comparison of the reacted reagent area against a whitebackground may aid in the detection of low levels of nitrite ion, whichmay otherwise be missed.

Availability: AMES REAGENT STRIPS for Urinalysis are available inbottles of 100 strips: MULTISTIX® 10 SG (#2300A); MULTISTIX® 9 (#2301A);MULTISTIX® 9 SG (#2303A); MULTISTIX® 8 SG (#2304A); MULTISTIX® 7(#2305A); N-MULTISTIX® SG (#2740A); MULTISTIX® SG (#2741A); N-MULTISTIX®(#2829A); MULTISTIX® (#2820A); and BILI-LABSTIX® (#2814A).

Any of these or other commercially available reagent test strips whichprovide pH, protein, nitrite, leukocyte esterase and preferably alsoeosinophil and/or TAME esterase activity data can be used according tothis disclosure to differentiate between bacterial infections, viralinfections and allergic conditions. Thus, in a fashion completelyanalogous to that described above for the Ames REAGENT STRIPS,commercially available reagent test strips produced by BoehringerMannheim Corporation may be used or adapted for this purpose. Forexample CHEMSTRIP 9, Catalog No. 417109, provides a readout forleukocytes, nitrite, pH, protein and several other analytes. Theinformation provided in the package insert for the CHEMSTRIP 6, 7, 8, 9,10 (which also provides a readout for specific gravity), is largelyanalogous to the information provided hereinabove from the Multistix®product. In our hands, testing of nasal secretions using the Boehringerproduct yielded results which, according to this invention, are similarto those obtained using the Multistix® product. Slight adjustments inthe color readouts and values thereof may be needed due to thedifferences between the color charts used by the two manufacturers, but,based on the instant disclosure, those skilled in the art are able tomake any necessary adjustments.

In one aspect of the invention, there is provided a reagent test stripspecifically adapted for rapidly, non-invasively and inexpensivelydifferentiating between allergic conditions and respiratory, viral andbacterial infections. In use, the test strip is optimized to provideinformation about nasal mucus secretions including, but not limited to,the pH, protein content, nitrite, leukocyte esterase activity and levelof eosinophil infiltration, such that all of the information presentedin FIG. 2 and a separate indication of the presence or absence ofallergy can be obtained from a single indicator strip. This is achieved,for example, by preparing a reagent test strip according to commerciallyavailable strips, but in addition, providing a means for measuring theamount of eosinophil cationic protein (ECP) or another eosinophilspecific protein or enzyme present in the nasal secretion, the presenceof which is proportional to the amount of eosinophil infiltration.Alternatively, the test strip could be made so as to selectively trapeosinophils, and the assay could then be for any substance found ineosinophils (such as an enzyme or any other detectable substance),without it being necessary for the substance to be specific foreosinophils. The novel reagent test strip of this invention, therefore,can include an indicator location on the strip comprising immobilizedeosinophil cationic protein (ECP) or other appropriate protein bound tolabeled anti-ECP antibody or other specific antibody. In this event, thestrip is contacted with mucus from a patient and all of the otherparameters, (pH, protein, nitrite, leukocyte esterase) are read from thestrip. The strip is then incubated for a sufficient amount of time sothat ECP present in the mucus, due to eosinophil infiltration, competesthe labeled antibody from the strip-bound ECP. As a result, uponvisualization of the label, the greater the amount of ECP present in themucus, the lower the amount of label visualized. The antibody could beenzymatically labeled, or labeled with biotin or avidin, which couldthen be visualized by methods well known in the art. Analogously, ECP orother eosinophil antigen specific antibody could be immobilized on thestrip which, upon exposure to nasal secretions, binds any ECP or othereosinophil specific protein or enzyme present in the secretion. Excesssample could then be washed from the strip and a second, labeled ECP orother eosinophil antigen specific antibody or chromogenic eosinophilspecific enzyme substrate could be contacted with the strip. In thiscase, the greater the signal upon development, the greater the amount ofeosinophilia in the patient. Preferably, an eosinophil specific enzymeis detected by providing a chromogenic reagent on the strip whichchanges color to an extent proportional to the amount of eosinophilspresent in the sample. In this context, the teachings of U.S. Pat. Nos.5,369,007 and 5,200,321, herein incorporated by reference, whiledirected to a very different art (detection of illicit drugs) areinstructive here. According to those patents, a microassay on a cardcould be adapted to the instant utility in either a displacement orcompetition mode, as described herein above, using eosinophil specificantigens and antibodies. Likewise, the concepts and methods disclosedtherein could be utilized to prepare the novel reagent test strip of theinstant invention. In yet another embodiment of the novel test strip ofthis invention, a portion of the strip contacted with the nasalsecretion is transparent. This portion is then simply stained andquantitated microscopically for eosinophils as described hereinabove,after reading all of the other reagent parameters. In addition,measuring the level of TAME esterase activity in the secretion providesa further indication of whether an allergic condition is present in agiven patient.

In any event, even with the use of a standard, commercially availablereagent test strip, all that is required for the method of thisinvention is that, in addition to contacting the nasal secretion with anappropriate reagent test strip and quantitation of the pH, nitrite,leukocyte esterase and protein, is that the mucus be evaluated foreosinophils. This is quickly and easily achieved by making a smear ofthe nasal secretion, staining the smear with eosine or other appropriatedye, and quantitating the number of eosinophils present per field.Alternatively, the hemocytometric method of Okuda et al. (Int ArchAllergy Immunol. [1994] 104:6) or of Settipane (Allergy Proc. [1985]6:303-366), herein incorporated by reference, could be used for thispurpose.

According to methods known in the art, nasal ECP has been found to rangebetween about 30 ng/ml in a “normal” individual, up to about 200 ng/mlin individuals experiencing acute allergic reactions (see Clin. Exp.Allergy, 1997, 27:270-276; see also JACI 1996, 97:104-112; see alsoClin. Exp. Allergy 1994, 24:1151-1156). Commercially availableantibodies for carrying out the method and for making the deviceaccording to the present invention may be obtained, for example, fromPharmingen. Thus, Pharmingen catalog number 15371A is a mouse IgG1monoclonal antibody (clone AHE-1) which recognizes human eosinophilperoxidase, an 81 kD granule protein specific to eosinophils. Pharmingencatalog number 15381A is a mouse IgG1 monoclonal antibody (clone AHE-2)which recognizes human eosinophil Major Basic Protein, a 14 kD granuleprotein specific to eosinophils. Accordingly, a wide variety ofeosinophil specific markers may be employed according to the method andfor the device of this invention. Thus, a protein selected from thegroup, but not limited to the group, eosinophil major basic protein(MBP), present at about 9 μg/million eosinophils, eosinophil cationicprotein (ECP), present at about 5 μg/million eosinophils, eosinophilderived neurotoxin (EDN), present at about 3 μg/million eosinophils, oreosinophil peroxidase (EPO), present at about 12 μg/million eosinophils,(see Textbook of Allergy, Principles and Practice, for concentrations ofthese markers), may be employed in the device or method according tothis invention, using commercially available antibody, or antibodydeveloped independently. As mentioned above, avidin/biotin conjugates ofsuch eosinophil specific markers may also be employed according to thisinvention.

Accordingly, this invention provides a method for rapidly,non-invasively and inexpensively differentiating between allergy andviral or bacterial respiratory infection. The method involves measuringthe pH, protein content, nitrite content, leukocyte esterase activityand eosinophil content or other substance found in the secretions ofallergic individuals, such as TAME esterase, of a sample of contactednasal secretion. A combination of a pH between about 7.5 and 9, amoderately strong presence of protein, and at least a trace of nitriteand leukocyte esterase as quantitated by the scoring method of FIG. 2indicates the presence of a bacterial infection. The combination of a pHbetween about 5.0 and 7.0, little or no protein, little or no nitrite,little or no leukocyte esterase activity (i.e. a low score on thescoring system) and moderate to strong eosinophil content or TAMEesterase activity is indicative of an allergic condition. Thecombination of a pH between about 5 and 7, little or no protein, nitriteor leukocyte esterase and the absence of eosinophil infiltrationindicates an upper respiratory tract viral infection. In a preferredembodiment of this method, each value obtained for pH, protein, nitrite,leukocyte esterase in a patient's nasal secretion is assigned a valuesuch that the sum of assigned values results in an enhancement in theclustering of patient data having bacterial sinusitis infections. Aseparate set of values is established to determine the presence orabsence of allergic rhinitis, based on eosinophils, their surrogatemarkers and/or other substances in nasal secretions of patientssuffering from allergic rhinitis, such as TAME esterase. This is not tosay the measurement has to be conducted separately. Actually, the TAMEesterase analysis or eosinophil concentration analysis or both may beconducted at the same time as the measurement of other parameters,either by using an appropriate dip-stick, collection device as disclosedherein, or by using pads of reagents sensitive to each test substance.However, the measurement of eosinophils or TAME esterase are not summedwith the remaining measurements. Those measurements are summedseparately according to the method of this invention, and the eosinophilor TAME esterase measurement is interpreted independently of the othertest results.

In a preferred embodiment, this method is practiced with a reagent teststrip. The test strips which are used in this testing may be thoseproduced by the Ames division of Miles laboratory as disclosed in any ofU.S. Pat. Nos. 3,438,737; 4,301,115; 4,637,979; 4,645,842; 4,657,855;4,704,460; 4,758,508, or by Boehringer Mannheim Corporation, asdisclosed in any of U.S. Pat. Nos. 3,359,072; 3,418,079; 3,712,853;3,897,214; 3,802,842; 4,013,416; 4,385,114. A reagent test stripspecifically adapted for rapidly, non-invasively and inexpensivelydifferentiating between allergy and respiratory infection is alsoprovided. This novel strip comprises reagents adapted to provideinformation on the pH, protein content, nitrite content, leukocyteesterase activity and eosinophil content or TAME esterase activity ofthe sample contacted. Absence of sinusitis and allergic rhinitis in thepresence of rhinorhea indicates the presence of a viral infection.

In a further aspect of the present invention, as disclosed withreference to FIG. 3, there is provided a novel collection device forhome or point of use care for the collection of nasal secretions and forconducting diagnosis therewith, without the need for end-user contactwith nasal secretion samples. In one embodiment, the collectionapparatus is a bag into which a patient blows their nose. In order toprevent the bag from bursting, in one embodiment, the bag comprises aseries of air vents located so as to permit air blown into the bag toescape, without at the same time permitting nasal secretions to escapefrom the container. This is most conveniently accomplished by designingthe collection device to fit snugly about the nose of a patient, suchthat upon blowing nasal secretions into the bag, the secretions aredeposited onto a bottom aspect of the bag, while air blown into the bagis permitted to escape from a series of holes disposed at a top aspectof the bag. This element of the invention is shown in FIG. 3, panel (1).Once the nasal secretion is deposited within the collection device, itmay be desirable to seal the holes, if present. This is accomplished byprovision of a piece of tape for this purpose. Alternative means forsealing the holes include provision of a flap element which may befolded over the holes, thereby sealing the holes; provision of acardboard, plastic or like sealing means, and equivalents thereof whichwould be suggested to those of ordinary skill in the art, based on thepresent disclosure. Thus, for example, in certain embodiments of thisinvention, a pre-crimped patient label is folded over with an adhesivestrip to ensure that none of the nasal secretion oozes out of the samplecontainer during handling. Naturally, the patient label and sealingmeans may also be provided as separate elements. Thus, a patient labelmay be affixed to the collection device, or the collection device may bemanufactured with a segment adapted to receive indelible patientidentification markings.

In addition, this embodiment of the invention shows a marginal aspect ofthe collection device as a sealable edge, such that after the nasalsecretions have been blown into the collection device, the nasalsecretions may be isolated from contact with external surfaces. The sealmay be created by any of a number of means known in the art, such as bymeans of a “zip-lock” means, wherein a first edge of the sealable margincomprises a strip of material which fits into a groove on the secondedge of the sealable margin, thereby sealing the contents within thecollection device. Alternatively, a fold-over sealing flap may seal themarginal edge of the collection device through which the patient insertstheir nose to deposit nasal secretions. The fold-over flap may itselfhave a pressure-sensitive adhesive strip disposed thereon, such thatupon folding the flap over the edge into which the nose is placed andthen removed, after deposit of nasal secretion, the flap may be made toseal on an external surface adjacent the opening. Alternate means forsealing the collection device may be employed without departing from thesubstance of this invention. Such modifications, equivalents orvariations of the sealing means as would be suggested to one of ordinaryskill in the art, based on the present disclosure, are thereforeincorporated herein.

Accordingly, with reference to FIG. 3, in one aspect of this invention,a bag-like collection device is provided which may be sealingly opened.The collection device is positioned over the nose of a patient and thenasal discharge is collected. Where an integral indicator device isincluded in the apparatus, the nasal secretion may be spread over theindicator device by external application of a spreading pressure.Appropriate time intervals are then observed, as disclosed herein, priorto reading the test results. In the event that nasal secretions arebeing collected from a child or infant, it will be appreciated that itmay be necessary to wipe or squeeze nasal secretion from the nasalpassage of the infant or child.

It should be appreciated by those skilled in the art that the embodimentof this invention shown in FIG. 3 is merely suggestive, and that thecollection device need not be limited to the specifics as shown therein.Thus, the indicator device may be contained in a separate container andinserted into the collection device, prior, concurrent with orsubsequent to collection of the nasal secretion. The nasal secretion,once collected and coded for a particular patient, may also be stored,either at ambient temperature, or in a cooled environment, until suchtime as the sample may be analyzed. At that time, the indicator devicemay be brought into contact with the nasal secretion to provide thedifferential readout as disclosed herein.

It will further be appreciated that the configuration of the collectiondevice may be subject to various modifications, as defined by suchpractical considerations as the desire to avoid patient embarrassment.One element that may assist in this regard is to provide a colored oropaque collection device. In this embodiment, the indicator device mayneed to be withdrawn from the collection device to permit accuratereading of the test results. Alternatively, the colored or opaqueelement may be affixed to the collection device in a detachable formatsuch that the coloration or opaque coating may be removed when theindicator device is to be read.

In a further embodiment of this invention, the collection device may beconfigured so as to minimize the possibility of reagent crosscontamination. For example, where a pH reagent is directly adjacent aprotein responsive reagent, it might be the case that buffer leachingfrom the pH sensitive reagent might negatively impact on the proteinconcentration reading, or vice versa. In order to minimize thispossibility, a series of vertical separations may be defined betweenadjacent pads of reactive reagents. In this manner, each reagent may beindividually contacted with nasal secretion, without the danger of crosscontamination of one reagent with another.

In yet a further embodiment of this invention, the reactive reagents,whether directly applied to a surface of the collection device or placedin the collection device in the form of a reagent strip, it may bebeneficial for the reactive reagents to be physically separated by aseparation means until such time as the analysis is to be conducted. Ifthe analysis is to be conducted right away, upon collection of nasalsecretion, the barrier may be removed or perforated, so that nasalsecretion may contact the reagent pads. If the analysis is to beconducted some time after collection of the nasal secretion, the barriermeans may be left intact, and removed only when the analysis is to beconducted. Alternative means for creating such a barrier include thepossibility of including a fold in the collection device, a zip-lockfeature, a breakable barrier or the like, separating the main chamber ofthe collection device from the reactive reagents.

It will also be appreciated that in order to facilitate manufacture ordistribution, the collection device may be formed on a continuous rollor the like. In this manner, multiple collection devices may bereleasably attached to each other. When the need arises for adifferential diagnosis to be made, one collection element isdisconnected from the remaining collection devices and is usedindividually. It will further be appreciated that the indicator elementsmay be combined into a single indicator element, and such indicatorelement may comprise an electronic means for data collection oranalysis.

It will further be suggested to those skilled in the art, based on thepresent disclosure, that a kit may be provided for conducting nasalsecretion analyses. Such a kit might include a sample collection device,such as the nasal secretion collection bag shown in FIG. 3. The kitmight further include either an integral means for providing thedifferential diagnostic readout disclosed herein, or a separate meansfor providing the differential diagnostic readout. Thus, a dipstickmight be included in the kit as a separate element, the dipstick may beincluded within the collection apparatus, or different chemicals asdefined herein may be affixed directly on an aspect of the collectionapparatus with which the collected nasal secretions may be brought incontact. Furthermore, it will be appreciated that the shape orconfiguration of the collection apparatus may be varied, withoutdeparting from the scope of this invention. Thus, round, square,elliptical or like shapes for the collection apparatus may becontemplated, in addition to the essentially trapezoidal shape of thedevice exemplified in FIG. 3.

ALTERNATIVE EMBODIMENT ELIMINATING NITRITE MEASUREMENT

In an alternative embodiment, the diagnostic methods taught above may bemodified such that the step of testing for nitrite content may beeliminated from the diagnostic system. The elimination of this step, aswell as the corresponding diagnostic reagents on a test strip, providesa more streamlined, less expensive, more efficient and equally effectivedetection of bacterial sinusitis. Thus, in a specific embodiment, thesubject invention pertains to a method that involves measuring the pH,protein content, and leukocyte esterase activity of a nasal secretionsample. A combination of a pH between about 7.5 and 9, a moderatelystrong presence of protein, and at least a trace of leukocyte esteraseas quantitated by the scoring method similar to that of FIG. 2 indicatesthe presence of a bacterial infection.

Thus, according to a one embodiment, the subject invention pertains to areagent test strip that comprises an indicator for determining pH, anindicator for indicating protein content, and an indicator forindicating leukocyte esterase activity. Further, the subject inventionpertains to a method of detecting bacterial sinusitis comprisingcollecting a nasal secretion sample; obtaining a diagnostic stripcomprising an indicator for determining pH, an indicator for determiningprotein content, and an indicator for determining leukocyte esteraseactivity; contacting said diagnostic strip with said nasal secretionsample; observing measurements of pH, protein content, and leukocyteesterase activity from said diagnostic strip. In a more preferredembodiment, a bacterial sinus infection is detected upon observing a pHof between about 7.5 and 9, a moderately strong presence of protein ormore, and leukocyte esterase activity.

Having generally described the present invention, including the bestmode for practicing the invention, reference is now made to the attachedclaims, which define the scope of exclusivity claimed herein.

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1. A method for detecting bacterial sinusitis, which comprises measuringa sample of a patient's nasal secretion for pH, protein concentration,and leukocyte esterase activity, wherein a pH between about 7.5 and 9, aprotein content of at least 100 mg/dL, and a presence of leukocyteesterase is indicative of bacterial sinusitis.
 2. The method of claim 1wherein said nasal secretion is collected in a sealable container. 3.The method of claim 1 wherein said container further comprises a unitaryreagent test strip.
 4. The method of claim 2 wherein a reagent teststrip is inserted into said container to obtain a differentialdiagnostic readout.
 5. The method according to claim 4 wherein saidcontainer comprises a series of holes disposed so as to permit air blowninto said container to escape, without at the same time permitting saidnasal secretion to escape.
 6. The method according to claim 5 whereinsaid container comprises a means for sealing said series of holes toprevent nasal secretion from oozing from said container.
 7. The methodaccording to claim 6 wherein said container comprises a means forassociating a particular nasal secretion sample with a particularpatient.